For an analysis of biochemical assays it is desired to detect several analytes simultaneously in so-called array arrangements. Such arrays are widely known on the basis of optical detection. It would advantageous to record electric measuring signals directly, and not indirectly over optical detection means, and to measure particle-tolerantly and independently of the volume. An electric detection would provide cost advantages and a tougher handling.
Starting out from classic electrode systems for electrochemical detection, considerable efforts have been made to miniaturize electrodes. The term usually designates electrochemically used electrode structures having dimensions of less than 5 μm. Wightman and Dipf describe possibilities of voltammetry at ultramicroelectrodes in Electroanalytical Chemistry, Ed. A. J. Bard (Marcel Dekker, New York 1988) vol. 15, p. 267.
Ultramicroelectrodes of this type also allow particular detection methods, such as redox recycling, compare Niwa et al., Electroanalysis 3(1991) 163–168, said method being particularly advantageously applied for biochemical affinity assays with enzyme marking, such as they are general practice in immuno and DNA assays. Electrode structures having a structural width of below 300 nm permit a marker-free detection of the affinity binding of large molecules to electrode-bound catcher molecules by using impedance spectroscopy, compare also DE-A 196 10 115.
A pair of planar interdigital electrodes for conductometric and voltammetric measurements is designated as array by Sheppard et al. in Anal. Chem., 65(1993) 1202.
Tang et al. Anal. Chimica Acta, 214 (1988) 187 suggest an individual interdigital electrode pair as a detector system for an immunoassay consisting of an antigen and an antibody.
However, all said arrangements have only been described with respect to an individual analyte determination, not allowing an individual electric detection of array-typical different molecule species.
Microelectrode arrays having 16 parallel strip electrodes and an electrode width of 0.1 mm have been described by Aoki et al., Anal. Chem. 64 (1992) 44 for an electrochemical detection. According thereto, different polarization voltages are applied at said individual strip electrodes and maintained constant. The electrodes are read out serially in msec cycles, without switches being allocated to said individual electrodes. A low-pass filter prevents the occurrence of charging currents. Said arrangement only provides a detection of individual or different electrode-active species in solution.
DE 4318519 describes an advancement of pairs of interdigital electrodes to an array having multiple interdigital electrodes for a simultaneous use at a multipotentiostat. According to said method, the potentials at said electrodes are individually controlled and kept constant. Said array is also only suited for a simultaneous and parallel measurement of an analyte in solution. Also having a multipotentiostat, 4 fields having dot-shaped microelectrodes in parallel for determining different metals by an anodic stripping method have been described, cf. DE 44 24 355 C2. The method only allows the particular stripping voltammetry as a detection method, voltage ramps having been modulated by a square-wave-voltammetry method.
The principle of a voltammetric parallel multi channel measurement at microelectrode arrays is described in Electroanalysis 8, 10 (1996) 891. Strein and Ewing, Analytical Chemistry 65 (1993) 1203 suggest an electrode array having embedded carbon fibers of a diameter of 1 to 2 μm. Neither of said methods allows a serial electric inquiry of different sensor positions.
Yon Hin et al., Sensors and Actuators B1 (1990) 550 describe a multianalyte electrode array comprising meander-shaped parallel electrode strips for a parallel analysis of glucose and galactose by a conductivity measurement. For this purpose, glucose oxidase and galactosidase are polymerized in conductive polypyrrole on the electrode surfaces, controlled by an electropolymerization. Said array using the electric conductivity as a detection quantity, indexing processes have no importance with respect to voltammetric malfunctions.
A nano-structured gold electrode array for immunodetection is described by Musiel et al., Journal of Vacuum Science and Technology B13 (6)(1995) 2781. Said electrode array is stochastically distributed by extracting nano particles from an insulating layer applied on gold and cannot be individually addressed and read out.
U.S. Pat. No. 5,605,662 describes an electrode array comprising individually controllable individual electrodes of a diameter of substantially 30 μm and larger counter electrodes separated therefrom on a silicon chip. Said array is not used for electrochemical detection, but only for an addressing and field production between individual electrodes coated with gel and counter-electrodes positioned at the edge of said array. By said produced field, charged molecules are transported to individual electrode positions or removed from said fields by a counter-polarization. A concrete case, for which said method is described, is the concentration of DNA in a gel above individual electrodes for a DNA hybridization at catchers, and in a reverse case, the elimination of mismatches by a field support of the DNA stringency treatment, cf. Sosnowski et al., Proc. Natl. Acad. Sci., USA, 94 (1997) 1119. The use of said system for molecular biological multianalyte diagnostic is described in U.S. Pat. No. 5,5632,957, the electric transport being combined with an optical detection.
An array for a potentiometric application, said array having more than thousand individually addressable electrode elements, is described by Hermes et al., Sensors and Actuators B 21(1994) 33. The individual positions of said sensor array are actively switched on only at the time of readout, whereas, in the non-readout state, no potential is supplied and no reaction takes place. CMOS switches for switching the electrodes on and off are individually arranged at each array position. A similarly structured multi electrode array comprising nMOS switches at each sensor position has been described by Fiaccabriono et al. in Sensors and Actuators B, 18–19 (1994) 675. In this type of arrays considerable charging currents are generated considerably affecting amperometric detection methods. In Anal. Chem. 66 (1994) 418, Kounaves et al. describe an array having 19 iridium electrodes of a diameter of 10 μm as individually addressable electrodes. Said electrodes were read out serially by a 2-electrode technology, and supplied with a potential only in the readout state.
A survey of electrochemistry at ultramicroelectrodes is to be found in Physical Electrochemistry, Ed. Rubinstein, Marcel Dekker, 1995 New York, pp. 131–208.
The application of a pair of 20–300 nm structured interdigital electrode arrays for a marker-free impedance analysis of a molecule conjugation on the electrode surfaces has been described in DE-A 196 10 115 (as above). An individual pair of nano-structured interdigital electrodes for admittance spectroscopy of dissolved molecules has been described in J. Vac. Sci. Technol. A 13 (3) (1995) 1755. A similar principle of impedance measurement in an electrode interspace of immobilized molecules by an interdigital pair of nanometer electrodes evaporated on a pit wall is shown in PCT/EP 96/05290. In all described impedance measurements using ultramicroelectrodes, an interdigital electrode pair was connected to a commercial impedance measuring device by two-pole technology.
A particular type of individually addressable sub-μm strip electrode arrays has been described by Nagale and Fritsch in Analytical Chemistry 70, 14 (1998) 2902 as stacked thin-film electrodes insulated with respect to each other. The cross-sections of the stacks have been used as active electrodes. A commercial computer-supported potentiostat comprising a working electrode, a reference electrode and a counter-electrode, has been used for electrochemical control. The 15 electrode layers have been read out serially by switching on and off.
A microelectrode array for extracellular activity measurement and stimulation of living cells and neuronal tissues uses individually addressable microelectrodes having a diameter of 14 μm, said microelectrodes being applied by a CMOS VLSI chip for stimulation and detection, each chip electrode being adapted to individually record cellularly generated biopotentials between 0.9–2.1 mV and 100–400 μV, cf. Pancrazio, et al. Biosensors & Bioelectronics 13(1998) 971. For said stimulation, frequencies between 0.7 and 50 kHz with bias potentials of 12–16 μV are applied. The electrodes are operated serially in a switched-on or switched-off state.
A method and an arrangement for enriching and cleaning molecules at large-area electrodes is described in PCT/DE 97/01368. According thereto, only small field strengths are generated and no detection methods are included.
A modification and coating of surfaces with biomolecules, such as they are used for the electric sensor array, is achieved by a covalent binding or adhesion to the metallic or non-metallic surfaces or to the walls of compartments. The molecules are applied as monolayers or multilayers by a covalent binding, by adsorption, by inclusion in polymers or as adhesive films, cf. Mandenius et al., Methods in Enzymology 137 (1988) 388. An adhesive layer production over cross-linked layers applied in gaseous or liquid phase, cf. Williams et al., Biosensors & Bioelectronics 9 (1994) 159, on surfaces with functionalized silanes as monolayers, cf. Fischer et al., Europhysics Letters 28 (2) (1994) 129–134, is widely known. To said silane derivatives, which may carry amino, thiol, aldehyde, hydroxyl, carboxyl or other functional groups, a very wide range of other compounds having suitable reactive groups are covalently bound by cross-linking methods, cf. Bäumert/Fasold, Methods in Enzymology, vol. 172, p. 584. In this manner, all bioactive substances suitable as affinity-binding catcher molecules, such as oligonucleotides, peptides, haptens and others are to be immobilized on the electrode surfaces.
A specific immobilization making use of the metal surface is the formation of self-assembling monolayers by thiol/gold bonds. After the formation of a self-assembling monolayer, an ordered binding of proteins, such as antibodies, is obtained e.g. over streptavidin/biotin couplings, cf. Spinke et al., Langmuir 9 (1993) 1821. In another preparation, histidine-marked proteins are orderly linked to the surfaces on gold surfaces via thioalkane chelating agents, cf. Krödger et al., Biosensors and Bioelectronics 14 (1999) 155.
A further method for selectively applying organic adhesive and coupling layers is the electropolymerization, for example for linking ferrocenes on platinum electrodes, cf. Karnau et al. in Anal. Chem. 66(1994) 994.
A number of methods for producing biomolecular arrays in microdimensions are customary. Putting miniaturized rings onto chip surfaces is derived from macroscopic stippling, said surfaces having been coated by immersion with corresponding molecules in advance, cf. [Rose, J. Ass. Lab. Autom. 3, 3 (1998) 53].
Blanchard in Genetic Engineering, Principles and Methods, 20 (1998) 111 succeeded in providing a piezoelectric printing method, similar to ink-jet printers, for structuring DNA-Chips.
The so-called micro contact printing, i.e. the transfer o molecules by microstamps has been described by Kumar and Whitesides, Appl. Phys. Lett. 63 (1993) 2002.
Mcgall et al., Proc. Natl. Acad. Sci., USA 93 (1996) 13555 suggested a solid phase synthesis on chip microareas, said synthesis permitting a nucleotide constitution by photoactivation.
According to U.S. Pat. No. 5,605,662, charged molecules are transported by electrochemical focusing from the solution to their binding positions in gels over electrodes.
Due to perforated membranes which are pressed onto chip surfaces, immobilization reactions at the open positions are possible on the surfaces in the liquid phase, cf. Ermantraut et al., Proc. of μTAS'98, Alberta, Can., 1998, p. 217.
The mentioned methods represent standard methods permitting an immobilization of DNA, oligonucleotides, proteins and other molecules at array positions.